CN115074356A

CN115074356A - New coronavirus nucleic acid extraction promoter and application thereof

Info

Publication number: CN115074356A
Application number: CN202210989492.6A
Authority: CN
Inventors: 刘恒贵; 孙建宏; 闫丽辉
Original assignee: Harbin Veterinary Research Institute of CAAS
Current assignee: Harbin Veterinary Research Institute of CAAS
Priority date: 2022-08-18
Filing date: 2022-08-18
Publication date: 2022-09-20

Abstract

The invention discloses a new coronavirus nucleic acid extraction promoter and application thereof, belonging to the technical field of biological detection. In order to solve the problems that the Ct value of the internal reference is reduced when the sample is placed for more than 24 hours and then extracted, and the Ct value of some internal references of the sample can not be even detected. The invention provides a new coronavirus nucleic acid extraction promoter, which comprises the following components: reducing agents, detergents and phosphate buffer solutions. The experimental result of adding the reagent into the sample collected by the new corona shows that the content of the nucleic acid of the internal reference and the new corona virus in the sample extracted by the paramagnetic particle method can be improved by more than 10 times. The nucleic acid extraction promoter is applied to new corona detection, can obviously improve the detection sensitivity of viruses and improve the detection rate of weak positive samples of new corona.

Description

New coronavirus nucleic acid extraction promoter and application thereof

Technical Field

The invention belongs to the technical field of biological detection, and particularly relates to a novel coronavirus nucleic acid extraction promoter and application thereof.

Background

In modern molecular biology experiments, the extraction of DNA and RNA from various sources is a necessary process for many experiments. Dnases and rnases are enzymes that specifically degrade nucleic acids, which are widely found in nature, so that nucleic acid molecules exposed to the outside are easily degraded, especially in tissue cells, and the contents of both enzymes are very high, and it is difficult to extract intact nucleic acid molecules from these materials if they are not properly handled. In intact virus particles, viral nucleic acids bind to various proteins, and enzymes that degrade DNA and RNA do not reach the nucleic acids, so the viral nucleic acids can be preserved intact. If viral nucleic acids are released from the viral particles, they degrade rapidly without protective measures, and therefore, the viral nucleic acids obtained in this case hardly guarantee the success of various molecular experiments, in particular, the detection experiments of viruses.

In the new crown test, samples are all from the pharynx or nasal cavity, and these samples not only contain a large amount of mucus, but also a swab scrapes off a large amount of epithelial cells during sampling. When these cells are disrupted, a large amount of RNase is released, and if these enzymes are not completely inactivated, the nucleic acid of the new coronaviruses is rapidly hydrolyzed after being released, in which case even a positive sample is difficult to detect in the fluorescent RT-PCR. Therefore, a certain amount of guanidine salt is added into the lysis solution in the new coronary detection tube at present as denaturant, and the denaturant can effectively inhibit the activity of the RNase within a certain time. However, in the process of detecting new coronas, the Ct value detected by the internal reference is often reduced if the sample is placed for more than 24 hours, and the Ct value cannot be detected even by some internal references. These phenomena indicate that the use of guanidinium salts alone as an inhibitor of rnases is not ideal and that a small amount of biologically active rnases are present after lysis of the sample in the lysis solution.

Disclosure of Invention

The invention aims to solve the problems that the Ct value of internal reference detection is reduced when the sample is placed for more than 24 hours and then extracted, and the Ct value of some internal references of the sample can not be detected.

The invention provides an accelerant for extracting nucleic acid of a novel coronavirus, which comprises the following components: reducing agent, detergent and phosphate buffer solution.

Further defined, the reducing agent is DTT.

Further defined, the detergent is SDS.

Further defined, the phosphate buffer is disodium hydrogen phosphate.

Further defined, the accelerator has the following components: DTT, SDS, and disodium phosphate.

Further defined, the accelerator has the following components: 0.1-10mM DTT, 0.5-5% SDS and 0.1-1M disodium hydrogen phosphate, pH 7.2.

Further defined, the accelerator has the following components: 10mM DTT, 5% SDS and 1M disodium phosphate, pH 7.2.

Further defined, the accelerator has the following components: 0.1mM DTT, 0.5% SDS and 0.1M disodium phosphate, pH 7.2.

The invention provides application of the accelerant in preparation of a kit for extracting the RNA of the new coronavirus.

The invention provides application of the promoter in improving the extraction efficiency of the nucleic acid of the new coronavirus.

Has the advantages that: the formula of the promoter for extracting the nucleic acid of the new coronavirus mainly comprises a reducing agent, a detergent and a phosphate buffer solution, wherein the reducing agent comprises DTT, and the detergent comprises SDS. The reducing agent is used at a final concentration of 0.1-10mM, preferably 1mM, and has the function of destroying disulfide bonds in RNase to permanently inactivate the RNase in the protective agent; the final use concentration of the detergent is between 0.5 and 5 percent, the recommended final use concentration is 0.5 percent, and the detergent has the functions of removing protein components in RNA, particularly effectively separating the relation between nucleic acid and protein in the extraction process of magnetic beads and promoting the combination of the nucleic acid and the magnetic beads; the final concentration of phosphate is 0.1-1M, the recommended final concentration is 0.1M, the function of the phosphate in the extraction promoter is to provide a stable buffer environment for nucleic acid, which is beneficial to stable storage of nucleic acid, and provide a salt ion environment in nucleic acid extraction, so that the magnetic beads extract RNA or DNA with stronger positive ions, and promote the electrostatic combination of nucleic acid and magnetic beads.

Drawings

FIG. 1 is the maximum dilution factor for positive test results;

FIG. 2 shows the maximum dilution factor at which the test results were positive.

Detailed Description

Example 1 nucleic acid extraction promoter of New coronavirus

Formulation of promoter for nucleic acid extraction of New coronavirus (10X) the experimental procedure was carried out as follows:

the formula of the extraction promoter comprises: DTT 10mM

SDS 5%

Disodium hydrogen phosphate: 1M

pH 7.2。

Comparative example 1 nucleic acid extraction promoter for New coronavirus

Extraction promoter control formula 1: SDS 5%

Disodium hydrogen phosphate: 1M

pH 7.2。

Comparative example 2 nucleic acid extraction promoter for New coronavirus

Extraction enhancer control 2 formulation: DTT 10mM

Disodium hydrogen phosphate: 1M

pH 7.2。

Comparative example 3 nucleic acid extraction promoter for New coronavirus

Extraction promoter control formula 3: DTT 10mM

SDS 5%

pH 7.2。

The experimental effect was verified using the following experiment:

the RNA extraction method of the novel coronavirus comprises the following steps:

1. 5 empty sample sampling tubes A1, A2, A3, A4 and B1 were taken, 1.8ml of a commercial sample preservation solution was added to each tube, 200. mu.l volumes of an extraction promoter (example 1), an extraction promoter (comparative example 2) and an extraction promoter (comparative example 3) were added to tubes A1 to A4, respectively, and 200. mu.l of pure water was added to a tube B1 as a control.

2. One tube of the sampling tube C containing 6ml of the sample preservation solution is taken, and the pharyngeal samples of two healthy people are collected by using a sampling swab. The collected pharyngeal swab is fully stirred in the preservation solution, so that the collected sample is fully released from the swab to the preservation solution.

3. 1ml of the sample in the tube C was added to the tube A1-A4 and the tube B1, respectively. Since the time from the sample collection to the sample sending to the detection laboratory for detection usually takes 1-4 hours, in order to simulate the process time, the sampling tubes A1-A4 and B1 to which the samples are added are placed at room temperature for 24 hours, and then 200ul of the samples are taken from A1-A4 and B1 respectively for magnetic bead method (the kit is purchased from Shuichi company) to extract the nucleic acids in the samples.

4. After the sample is extracted in the automatic extraction instrument, 5 mu l of the sample is taken from the eluent for fluorescence RT-PCR detection, and the detection kit is a novel coronavirus 2019-nCoV nucleic acid detection kit (fluorescence PCR method) purchased from Wuhanmingde company. The effect of the detection was evaluated by the Ct value of the internal reference RNase-P gene detected in the sample.

The results show that the Ct values for the a1 sample group with extraction enhancer added are reduced by approximately 4.80%, 4.86%, 2.67%, and 10.36% compared to the extraction enhancer added controls a2, A3, a4, and the control B1 without any extraction enhancer, respectively. The results are shown in table 1, and various components in the formula of the extraction promoter have the function of obviously reducing the Ct value of nucleic acid for nucleic acid detection, so that the content of the extracted nucleic acid is obviously improved. The extracted samples are further diluted by 10-fold serial dilution or 2-fold specific dilution, and the results show that the nucleic acid extracted from the sample A1 can be detected to be positive when diluted by 1 ten thousand times, but the nucleic acids extracted from the control samples A2, A3, A4 and B1 can be detected to be positive only when diluted by 4000-fold, 2000-fold and 1000-fold (as shown in FIG. 1), and the nucleic acids extracted from the control samples A2, A3, A4 and B1 are negative when further diluted. These results show that the nucleic acids extracted by the samples to which the extraction promoter was added were improved by 10 times or more as compared with the samples to which no extraction promoter was added, and by 2.5 times, 2.5 times and 5 times or more as compared with the samples A2, A3 and A4 to which the respective extraction promoter controls were added (as shown in Table 2). These results clearly show that the extraction promoter can effectively improve the efficiency of RNA detection in a sample, and various components have the function of improving the nucleic acid extraction efficiency to different degrees, and have positive synergistic effect on the nucleic acid extraction effect.

TABLE 1

Note: the sensitivity is the ratio of the maximum dilution times of the samples which are detected to be positive, the positive is the Ct value, and the negative is the Ct value.

Table 2: the detection sensitivity of the A1 group is improved by multiple compared with that of other control groups

The following experimental procedure was performed by applying the prepared extraction promoters and various extraction promoter controls to the extraction of new coronavirus nucleic acids, and the new coronavirus particles were replaced with the pseudoviruses we prepared. The specific operation and experimental results are as follows:

1. 5 empty sample sampling tubes C1, C2, C3, C4 and D1 are taken, 1.8ml of commercial sample preservation solution is added into each tube, then 200 microliter volumes of extraction accelerator, extraction accelerator control 1, extraction accelerator control 2 and extraction accelerator control 3 are respectively added into the sampling tubes C1 to C4, and 200ul volumes of pure water are added into the tube D1 as controls.

2. One tube of the sampling tube E containing 6ml of the sample preservation solution is taken, and pharyngeal samples of two healthy people are collected by using a sampling swab. The collected throat swab was added to the storage solution while stirring thoroughly, 200ul of pseudovirus solution was added, and after thoroughly mixing, 1ml of the liquid in tube E was added to tubes C1, C2, C3, C4 and D1, respectively.

3. Since the time from the sample collection to the sample transfer to the detection laboratory typically takes 1-4 hours, to simulate this process time, all the sampling tubes to which the samples were added were first left at room temperature for 24 hours, and then 200ul of each tube was individually removed for extraction of nucleic acid from the samples by the magnetic bead method (kit available from major).

4. After the sample is extracted in the automatic extraction instrument, 5 mu l of the sample is taken from the eluent for fluorescence RT-PCR detection, and the detection kit is purchased from Wuhanmingde company. The effect of the detection was evaluated by Ct values of the N gene of the novel coronavirus and the ORF1ab gene in the sample.

The results showed (as tables 3 and 4 below) that in sample C1 with the addition of extraction enhancer, the Ct values of the N gene and ORF1ab gene were reduced by 14.93%, 6.34%, 5.59%, and 2.1% on average over C2, C3, C4 of the extraction enhancer control group and D1 of the control group without any extraction enhancer, and the Ct values of the ORF1ab gene were reduced by 13.34%, 7.41%, 6%, and 2.35% on average over C2, C3, C4 of the extraction enhancer control group and D1 of the control group without any extraction enhancer, respectively.

The extracted new coronaviral nucleic acid was diluted 10-fold or 2-fold, and further analyzed for changes in the content of extracted viral nucleic acid, the results showed that the maximum dilution fold for positive detection of the nucleic acids N and ORF1ab genes from samples C1, C2, C3, C4 and D1 were 12000, 4000, 2000 and 1000-fold, respectively (as shown in fig. 2), and the nucleic acids extracted from C1 group to which the extraction promoter was added were 3-fold, 6-fold and 12-fold higher than those extracted from C2, C3, C4 and D1 group to which no extraction promoter was added, respectively (as shown in table 5). These results indicate that the nucleic acid extraction promoter can effectively improve the detection efficiency of the novel coronavirus nucleic acid in the sample. And various components have the function of improving the extraction efficiency of the new coronavirus nucleic acid to different degrees, and the components have positive synergistic effect on the extraction effect of the coronavirus nucleic acid.

TABLE 3

Claims

1. An accelerant for extracting nucleic acid of a new coronavirus, which is characterized by comprising the following components: reducing agents, detergents and phosphate buffer solutions; the reducing agent is DTT; the detergent is SDS.

2. The enhancer of claim 1, wherein the phosphate buffer is disodium hydrogen phosphate.

3. Accelerator according to claim 1, characterized in that the composition of the accelerator is as follows: 0.1-10mM DTT, 0.5-5% SDS and 0.1-1M disodium hydrogen phosphate, pH 7.2.

4. Accelerator according to claim 1, characterized in that the composition of the accelerator is as follows: 10mM DTT, 5% SDS by mass and 1M disodium hydrogen phosphate, pH 7.2.

5. Accelerator according to claim 1, characterized in that the composition of the accelerator is as follows: 0.1mM DTT, 0.5% SDS by mass and 0.1M disodium hydrogen phosphate, pH 7.2.

6. Use of the promoter of any one of claims 1 to 5 for the preparation of a kit for the extraction of new coronavirus RNA.

7. Use of the promoter according to any one of claims 1 to 5 for increasing the efficiency of extraction of nucleic acid from a novel coronavirus.